1. Field of the Invention
The present invention relates to a device and method for measuring physicochemical changes in a biological sample, typically a cell, and an apparatus comprising the device. More particularly, the present invention relates to a device and method for measuring physicochemical changes in a cell correlating with a macroscopic level of ion channel activity of the whole cell, and an apparatus comprising the device. The present invention also relates to a method and device for screening drugs.
2. Description of the Related Art
Conventionally, physical signals or chemical signals emitted in association with the activity of a biological sample, such as a cell, are measured by a measurement apparatus for capturing changes in an electrical signal, the fluorescence intensity of a fluorescence indicator taken up by a biological sample, or the like, as a digital signal.
For example, when the ion channel activity of a cell is measured, a level of a single ion channel is measured by an electrophysiological measurement apparatus using a microelectrode probe (e.g., patch clamp) and a specialized control apparatus as follows. The apparatus captures the amount of electricity passing through an ion channel of a cell as a digital signal and, based on amount of electricity, calculates the duration, timing, number of times, and the like of opening or closing of the ion channel. In this technique, a microelectrode probe is inserted into a cell and electric current passing through a biological sample is measured. The technique is therefore called an intracellular recording method.
In a patch clamp technique, a small portion (patch) of cell membrane is attached to a tip portion of a micropipette with a microelectrode probe, and is used to electrically record ion transportation through a single ion channel protein. The patch clamp technique is one of a few number of cell biological techniques which can be used to investigate the function of a single protein in real time (see, e.g., Molecular Biology of the Cell, 3rd Ed., Garland Publishing, Inc., New York, 1994, Japanese Version, translation supervised by Keiko Nakamura et al., pp. 181-182, 1995, Kyoikusha).
The ion channel activity of a whole cell can also be measured by a fluorescence measurement method capturing the amount of ions flowing into the cell as a digital signal.
In a fluorescence measurement technique, a light emitting indicator or a fluorescent pigment which emit light in accordance with the concentration of a specific ion, is combined with an up-to-date image processing method (e.g., a fluorescence image of a cell is captured by a CCD camera or the like, and the movement of ions in the cell is monitored) to measure the electrical activity of the entire cell.
The patch clamp technique requires special techniques for preparation, manipulation and the like of a micropipette, and much time for measuring one sample. Therefore, the patch clamp technique is not suitable for screening a large quantity of candidate compounds for a drug at high speed. The fluorescence measurement technique can screen a large quantity of candidate compounds for a drug at high speed. However, the fluorescence measurement technique requires a step of staining a cell. During measurement, pigments cause high background noise, and the fluorescence intensity decreases with time, resulting in poor signal to noise ratio (S/N).
Another technique for observing an electrochemical change in a biological sample, is disclosed in JP No. 2949845, U.S. Pat. Nos. 5,810,725, 5,563,067, Japanese Laid-Open Publication No. 9-827318, WO 01/25769 A2, U.S. Pat. No. 5,187,069, WO 98/54294, WO 99/66329, WO 99/31503, and the like, in which a substrate provided with multiple electrodes is employed.
JP No. 2949845, U.S. Pat. Nos. 5,810,725, 5,563,067, and Japanese Laid-Open Publication No. 9-827318 disclose an integrated multiple electrode comprising microelectrodes prepared by photolithography on a glass substrate and capable of measuring electrical changes in cells, and a measurement system using the same.
WO 01/25769 A2 discloses a substrate in which an insulating substrate provided with through holes and a biological sample, such as a cell containing an ion channel, is placed on the through holes so that a gigaseal is provided on the surface of the insulating substrate including the cell; a reference electrode and a measuring electrode, which are provided in two respective domains separated by the gigaseal, can be used to measure electric current generated by ions passing through an ion channel of the cell.
U.S. Pat. No. 5,187,069 discloses a device capable of monitoring the growth of cells by culturing the cells on electrodes and measuring impedance changes.
WO 98/54294 discloses a device in which cells are adhered onto a planar electrode and an electrical signal thereof is measured.
WO 99/66329 discloses a device for observing the activity of cells on a porous material by measuring resistance or impedance changes, and an assay using the same.
WO 99/31503 discloses a method in which a substrate provided with through holes is employed, patch clamps are established by trapping cells with the through holes, and changes in electric current are measured.
Any of the above-described conventional techniques are characterized in that the electrical activity of cells is determined on a planar electrode, and small through holes are provided in an insulating substrate so that patch clamp is formed with cells and, the substrate, whereby electric current generated by ions passing through an ion channel can be monitored. However, when a planar electrode is employed, a signal emitted from a biological sample leaks into solution so that the sensitivity of the measurement is disadvantageously reduced. In the method in which small through holes are provided in an insulating substrate so that patch clamps are formed with cells and the holes, the possibility of forming gigaseal patch clamps is low. Moreover, the cell membrane must be destroyed or physically injured to form patch clamp, so that it is not possible to measure the activity of intact cells. Patch clamp formation techniques also have a difficulty in adjusting suctioning pressure for a biological sample. Multiple channels would impractically require multiple pressure adjusting mechanisms. In these regards, conventional planar electrodes are suitable for high-speed drug screening but have poor sensitivity. The insulating substrate having small through holes is not suitable for high-speed drug screening. For similar reasons, an automated patch clamping robot requires much time for sample processing and is not suitable for high-speed drug screening.